Biocompatible phase invertible proteinaceous compositions and methods for making and using the same

ABSTRACT

Biocompatible phase invertible proteinaceous compositions and methods for making and using the same are provided. The subject phase invertible compositions are prepared by combining a crosslinker and a proteinaceous substrate. The proteinaceous substrate includes one or more proteins and a polyamine, where the polyamine and a proteinaceous substrate are present in synergistic viscosity enhancing amounts, and may also include one or more of: a carbohydrate, a tackifying agent, a plasticizer, or other modification agent. In certain embodiments, the crosslinker is a heat-treated dialdehyde, e.g., heat-treated glutaraldehyde. Also provided are kits for use in preparing the subject compositions. The subject compositions, kits and systems find use in a variety of different applications.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.14/178,093, filed Feb. 11, 2014, which is a continuation of U.S. patentapplication Ser. No. 12/757,432, filed Apr. 9, 2010, which claims thebenefit of priority of U.S. Provisional Patent Application No.61/170,545, filed on Apr. 17, 2009, which are incorporated herein byreference.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The field of biocompatible phase invertible proteinaceous compositions,and methods for making and using the same.

A number of sealant compositions have been used to control fluid leakageat a surgical site, as well as for other applications. However,currently available sealant compositions may suffer from seriouslimitations with regards to the field in which they can be used, as wellas their biocompatibility and their physical properties. Side effects,such as inflammation, acute fibrous formation at the wound site,toxicity, inability to be used in a bloody field, poor physicalproperties of the sealant, and poor adhesion to the surgical site, mayhave a serious impact on the patient and resultantly may play asignificant role in the long term efficacy of the repair. Further,useful sealants have properties that can render them more effective forsurgical application. Characteristics, such as the ability to belocalized to a specific location, adequately long or shortpolymerization times, and adequate in vivo resorption characteristics,are vital to a successful completion of the sealing procedure.

As such, there is a continued need for the development of newbiocompatible compositions for use as sealants, as well as for use inother applications.

2. Background Art

Various phase invertible compositions and applications are reported inU.S. Pat. Nos. 3,438,374; 5,292,362; 5,385,606; 5,575,815; 5,583,114;5,843,156; 6,162,241; 6,290,729; 6,310,036; 6,329,337; 6,371,975;6,372,229; 6,423,333; and 6,458,147; as well as U.S. Application Nos.:2002/0015724; 2002/0022588; 2002/0183244; and 2004/0081676.

BRIEF SUMMARY OF THE INVENTION

Biocompatible phase invertible proteinaceous compositions and methodsfor making and using the same are provided. The subject phase invertiblecompositions are prepared by combining a crosslinker and a substrate.The substrate includes a proteinaceous material and a polyamine in aweight ratio having a synergistic viscosity enhancing effect on thesubstrate composition, and at least in many embodiments, the substrateincludes a carbohydrate. The proteinaceous material includes one or moreproteins, such as an albumin protein. The polyamine is a cationicpolymeric amine, such as a polyethylenimine. In certain embodiments, thecrosslinker is a heat-treated dialdehyde, such as heat-treatedglutaraldehyde. In certain embodiments, the crosslinker is amacromolecular crosslinking agent, such as a heat-treated dialdehydecrosslinked to a physiologically acceptable polymer, such as aglycosaminoglycan. In certain embodiments, the kinematic viscosity ofthe substrate and the crosslinker are approximately the same. Alsoprovided are methods of production and kits for use in preparing thesubject compositions. The subject compositions, methods, kits andsystems find use in a variety of different applications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the synergistic effect of polyethyleneimine (PEI) onthe kinematic viscosity of various biocompatible phase invertiblecompositions according to representative embodiments of the subjectinvention.

DETAILED DESCRIPTION OF THE INVENTION

Biocompatible phase invertible proteinaceous compositions and methodsfor making and using the same are provided. The subject phase invertiblecompositions are prepared by combining a crosslinker and a substrate,where the substrate includes a proteinaceous material and a polyamine ina weight ratio having a synergistic viscosity enhancing effect on thesubstrate composition. At particular weight ratios of polyamine toproteinaceous material, the unexpected synergistic effect of increasingthe kinematic viscosity of the substrate significantly decreases theflow rate from the site of application to a desired level, whilepromoting efficient mixing and curing of the combined crosslinker andsubstrate compositions, thereby increasing the utility of thebiocompatible phase invertible proteinaceous compositions. Also providedare kits for use in preparing the subject compositions. The subjectcompositions, kits and systems find use in a variety of differentapplications.

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

In further describing the subject invention, the subject phaseinvertible compositions are described first in greater detail, followedby a review of representative applications in which the compositionsfind use, as well as a review of kits and systems that find use inmaking or using the subject phase invertible compositions.

Biocompatible Phase Invertible Proteinaceous Composition.

As summarized above, the subject invention provides a biocompatiblephase invertible proteinaceous composition that, over time, undergoes aphase inversion from a first, fluid state to a second, solid state. Thesubject phase invertible compositions are characterized by being capableof bonding tissue in both wet (e.g., blood) and dry environments, whereadhesion of the composition to the tissue is exceptionally strong. Afurther feature of the subject compositions is that they are welltolerated and do not elicit a substantial inflammatory response, if anyinflammatory response.

The subject phase invertible proteinaceous compositions are prepared bycombining or mixing a proteinaceous substrate with a crosslinker. Eachof these precursor components or compositions is now reviewed separatelyin greater detail.

Proteinaceous Substrate.

The substrate from which the subject phase invertible compositions areprepared is a proteinaceous substrate, and is generally a fluidcomposition, e.g., an aqueous composition, made up of at least aproteinaceous component and a polyamine component, and in manyembodiments, a carbohydrate component. The proteinaceous substrate mayfurther include one or more additional components, including, but notlimited to: a tackifying agent; a plasticizer; and the like.

The proteinaceous component and polyamine component are present in aweight ratio that provides a synergistic viscosity enhancing effect onthe proteinaceous substrate. By “synergistic viscosity enhancing effect”is meant a viscosity enhancing effect produced by the interaction of twoor more components that is greater than the sum of the viscosity effectproduced by each component in the absence of the other. Typically, theproteinaceous and polyamine components are present in a weight ratio ofpolyamine to proteinaceous material from about 1:4 to 1:400. Certainratios of polyamine to proteinaceous material result in a substratecomposition which exhibits synergistic viscosity effects of particularinterest. For example, in a phase invertible composition comprisingsynergistic viscosity enhancing amounts of proteinaceous material, suchas albumin, and a highly cationic polyamine, such as polyethylenimine,the weight ratio of the polyamine to proteinaceous material is generallyfrom about 1:5 to 1:100, usually from about 1:8 to 1:80, and moreusually from about 1:10 to 1:40.

In many embodiments, when a carbohydrate is included in theproteinaceous substrate, the carbohydrate and proteinaceous componentsare present in a weight ratio of carbohydrate to proteinaceous materialfrom about 1:8 to 1:800. Certain ratios of carbohydrate to proteinaceousmaterial result in a substrate composition of specific interest. Forexample, in a phase invertible composition comprising synergisticviscosity enhancing amounts of proteinaceous material, such as albumin,and a highly cationic polyamine, such as polyethylenimine, the weightratio of the carbohydrate to proteinaceous material is about 1:130 to1:400, usually about 1:150 to 1:350, and in certain embodiments, about1:200 to 1:300.

Proteinaceous Component.

The proteinaceous component of the substrate is made up of one or moredistinct proteins. The proteins of this component may be eithersynthetic or naturally occurring proteins, where the proteins may beobtained/prepared using any convenient protocol, e.g., purification fromnaturally occurring sources, recombinant production, syntheticproduction, and the like, where in certain embodiments the proteins areobtained from naturally occurring, e.g., bovine or human, sources.Specific proteins of interest include, but are not limited to: albumins,collagens, elastins, fibrins, and the like.

The amount of protein in the substrate composition may vary, where thespecific selection of concentration is dependent on the desiredapplication and product parameters desired therefore, such as tenacity,hardness, elasticity, resorption characteristics and plateletaggregation effects. In certain embodiments, the total protein totalconcentration in the substrate compositions ranges from about 1 to 75%(w/w), such as 1-50% (w/w), including 5 to 40% (w/w).

In certain embodiments, the primary protein of the substrate compositionof this embodiment is albumin, where the albumin may be a naturallyoccurring albumin, e.g., human albumin, bovine albumin, etc., or avariant thereof. As is known in the art, the albumin may be purchased inpowdered form and then solubilized into an aqueous suspension, oralternately, may be purchased in aqueous form. Purified albumin mayderived from any one of a number of different sources including, bovine,ovine, equine, human, or avian in accordance to well known methods(e.g., Cohn et. Al, J. Amer. Chem. Soc. 69:1753) or may be purchased inpurified form from a supplier, such as Aldrich Chemical (St. Louis,Mo.), in lyophilized or aqueous form. The albumin may be derivatized toact as a carrier for drugs, such as heparin sulfate, growth factors,antibiotics, or may be modified in an effort to moderate viscosity, orhydrophilicity. Derivitization using acylating agents, such as, but notlimited to, succinic anhydride, and lauryl chlorides, are useful for theproduction of binding sites for the addition of useful molecules. Inthese embodiments where the proteinaceous component includes albumin,the albumin may be present in concentrations ranging from about 10 toabout 50% (w/w), such as from about 30 to about 40% (w/w).

In certain embodiments, the proteinaceous component also includes acollagen, e.g., a naturally occurring collagen (human, bovine) orsynthetic variant thereof. In accordance with the invention, thecollagen may be in dry or aqueous forms when mixed with the albumin.Collagen may be derivatized to increase it utility. Acylating agents,such as anhydrides or acid chlorides, have been found to produce usefulsites for binding of molecules such as growth factors, and antibiotics.When present, the collagen sometimes ranges from about 1 to about 20%(w/w), including from about 1 to about 10% (w/w), such as from about 1to about 4% (w/w), including from about 2 to 4% (w/w).

The subject proteinaceous component, as described above, may or may notinclude one or more active agents, e.g., drugs, present in it, asdesired. When present, the agent(s) may be bound to the polymers, asdesired.

Polyamine Component.

The polyamine component of the substrate is a cationic polymeric amine.Polyamines can be linear or branched, substituted or unsubstituted, andinclude homopolymers, heteropolymers and copolymers of cationicpolyalkylenimines, polyvinylimines, polyamino acids, and the like. Themolecular weight of such polymers is generally greater than about 500Daltons, having an average molecular weight ranging from about 500 to 1million Daltons, and usually from about 500 to about 100,000 Daltons.Typically, the polyamine component of the substrate is a highly cationicpolymeric amine, with a high charge density that allows it to absorbtightly on negatively charged substances. Examples of such polyaminesinclude, but are not limited to, polyethylenimines (PEI),polyvinylamines (PVA), polylysine (PL), and derivatives thereof, such aspolylysine copolymers, polyvinylamine copolymers,carboxylated-polyethylenimine, a phosphorylated-polyethylenimine, asulfonated-polyethylenimine, an acylated-polyethylenimine, hydroxylatedwater-soluble polyethylenimines, and the like. Polyamines arecommercially available, such as from Sigma-Aldrich (St. Louis, Mo., USA)or BASF (Ludwigshafen, Germany), and include linear or branched,substituted or unsubstituted PEIs, PVAs, and PLs, such as theLupasol.RTM. PEI product series from BASF.

The polyamines may optionally include degradable bonds, for example,branched PEI crosslinked with degradable ester bonds is an example of aPEI derivative of this'type. The polyamine may also be derivatized toact as a carrier for drugs, such as heparin sulfate, growth factors,antibiotics, or may be modified in an effort to moderate viscosity, orhydrophilicity. Derivatization using acylating agents, such as, but notlimited to, succinic anhydride, acyl chlorides, and lauryl chlorides,are useful for the production of binding sites for the addition ofuseful molecules. Polyamines of particular interest are branched, suchas a branched polyamine having at least one primary, at least onesecondary, and at least one tertiary amine group, such as a branchedPEI.

In certain embodiments, the polyamine component of the substratecomposition is PEI, where the PEI may be linear, branched, substitutedor unsubstituted, or mixtures thereof. As is known in the art, the PEImay be synthesized in accordance to well known methods (e.g.,“Encyclopedia of polymer science and technology,” Jacqueline I.Kroschwitz, H. F. (Herman Francis) Mark, Wiley-Interscience, 2004;Murata et al., J Biochem. (2008) 144(4):447-455; Ito et al., J ControlRelease (2006) 112(3):382-388; Brus et al., Eur J Pharm Biopharm. (2004)57(3):427-430; Forrest et al., Bioconjug Chem. (2003) 14(5):934-940;Petersen et al., Bioconjug Chem. (2002) 13(4):812-821; and Sagara etal., J Control Release. (2002) 79(1-3):271-281; and U.S. Pat. Nos.4,187,256; 5,990,224; and 6,652,886) or may be purchased from asupplier, such as Sigma-Aldrich or BASF. Of specific interest isbranched PEI containing a mixture of primary, secondary and tertiaryamines, and having a molecular weight of about 50,000 to 100,000, suchas PEI (catalogue number 195444) available from MP Biomedical, Inc.

The amount of polyamine in the substrate composition may vary, where thespecific selection of concentration is dependent on the desiredapplication and product parameters desired, such as tenacity, hardness,elasticity, resorption characteristics and platelet aggregation effects.The polyamine sometimes ranges from about 0.1 to about 10% (w/w),including from about 0.1 to about 4% (w/w), such as from about 0.5 toabout 4% (w/w), including from about 0.5% to 3% (w/w), from about 1 to3% (w/w), and from about 1 to 2% (w/w).

As discussed above, certain ratios of polyamine to proteinaceousmaterial result in a substrate composition which exhibits synergisticviscosity effects, and in many embodiments, a carbohydrate component isincluded as well. As such, the weight percent of polyamine in thesubstrate composition is selected so as achieve the desired ratio andsynergistic viscosity effect, such as described in the Experimentalsection.

The subject polyamine component, as described above, may or may notinclude one or more active agents, e.g., drugs, present in it, asdesired. When present, the agent(s) may be bound to the polymers, asdesired.

Optional Components.

The above described substrate component of the subject compositions may,in certain embodiments, include one or more optional components thatmodify the properties of the phase invertible composition produced fromthe substrate and crosslinker Representative optional components ofinterest are now discussed in greater detail below.

Tackifying Agent.

Also present may be one or more tackifying agents. Tackifying agentsimprove the adhesiveness of the sealant to the biological surface. Inmany embodiments, the adhesion modifiers are polymeric compounds havingcharged functionalities, e.g., amines, etc. Whereas numerous adhesionmodifiers may be used, one of particular applicability is PEI, whichaids attachment to biological surfaces. In addition, the presence of PEIin the substrate significantly enhances the presence of amine terminalssuitable to produce crosslinks with the crosslinking agent. When PEI isincluded as both a tackifying agent and polyamine component, the ratioof proteinaceous material to PEI in the substrate composition isselected so as to exhibit the desired synergistic viscosity effect, suchas described in the Experimental section. Additional adhesion modifiersof interest include, but are not limited to: gelatin,carboxymethylcellulose, butylhydroxytoluene, etc.

In certain embodiments of the invention, tackifying agents are used tomodify adhesion to the biological substrate while simultaneouslycreating a procoagulant. In certain embodiments, the tacking agents arepresent in concentrations of from about 0.1 to about 10% (w/w), such asfrom about 0.1 to about 4% (w/w).

Plasticizing Agents.

In accordance to the invention, a plasticizing agent may be present inthe substrate. The plasticizing agent provides a number of functions,including wetting of a surface, or alternately, increasing the elasticmodulus of the material, or further still, aiding in the mixing andapplication of the material. Numerous plasticizing agents exist,including fatty acids, e.g., oleic acid, palmitic acid, etc.,dioctylphtalate, phospholipids, and phosphatidic acid. Becauseplasticizers are typically water insoluble organic substances and arenot readily miscible with water, it is sometimes advantageous to modifytheir miscibility with water, by pre-mixing the appropriate plasticizerwith an alcohol to reduce the surface tension associated with thesolution. To this end, any alcohol may be used. In one representativeembodiment of this invention, oleic acid is mixed with ethanol to form a50% (w/w) solution and this solution then is used to plasticize theproteinaceous substrate during the formulation process. Whereas the typeand concentration of the plasticizing agent is dependent upon theapplication, in certain embodiments the final concentration of theplasticizing agent is from about 0.01 to 10% (w/w), including from about2 to about 4% (w/w). Other plasticizing agents of interest include, butare not limited to: polyethylene glycol, glycerine, butylhydroxytoluene,etc.

Carbohydrate Procoagulant.

In certain embodiments, the substrates include a carbohydrateprocoagulant. Chitosan and derivates of chitosan are potent coagulatorsof blood and, therefore, are beneficial in formulating sealant materialscapable of sealing vascular injuries. While virtually all chitinmaterials have been demonstrated to have some procoagulant activity, inaccordance to the invention, the use of acetylated chitin is employed asan additive for the formulation of sealant intended for blood control.Acetylation of the molecule can be achieved in a number of differentways, but one common method is the treatment of chitosan/acetic acidmixtures with acid anhydrides, such as succinic. This reaction isreadily carried out at room temperature. In accordance with theinvention, gels created in this manner combined with proteinaceoussubstrates and crosslinked in situ are beneficial for the creation of abiocomposite structural member. As such, the carbohydrate procoagulantmay be chitosan, low molecular weight chitosan, chitin, chitosanoligosaccharides, and chitosan derivatives thereof. In accordance withthe teachings of this invention the carbohydrate component, e.g.,chitosan, may be present in concentrations ranging from about 0 to about20%, such as from about 0.1 to about 5% (w/w).

Fillers.

Fillers of interest include both reinforcing and non-reinforcingfillers. Reinforcing fillers may be included, such as chopped fibroussilk, polyester, PTFE, NYLON, carbon fibers, polypropylene,polyurethane, glass, etc. Fibers can be modified, e.g., as describedabove for the other components, as desired, e.g., to increasewettability, mixability, etc. Reinforcing fillers may be present fromabout 0 to 40%, such as from about 10 to about 30%. Non-reinforcingfillers may also be included, e.g., clay, mica, hydroxyapatite, calciumsulfate, bone chips, etc. Where desired, these fillers may also bemodified, e.g., as described above. Non-reinforcing fillers may bepresent from about 0 to 40%, such as from about 10 to about 30%.

Biologically Active Agents.

Biologically active agents may be included, e.g., bone growth factors,tissue activators, cartilage growth activators, small molecule activeagents, etc. Thus, the biologically active agents can include peptides,polypeptides, proteins, saccharides, polysaccharides and carbohydrates,nucleic acids, and small molecule organic and inorganic materials.Specific biologically active agents include antibiotics, antivirals,steroidal and non-steroidal anti-inflammatories, antineoplastics,anti-spasmodics including channel blockers, modulators ofcell-extracellular matrix interactions including cell growth inhibitorsand anti-adhesion molecules, enzymes and enzyme inhibitors,anticoagulants, growth factors, DNA, RNA and protein synthesisinhibitors, anti-cell migratory agents, vasodilators, and other drugsused for treatment of injury to tissue. Examples of these compoundsinclude angiotensin converting enzyme inhibitors, anti-thromboticagents, prostacyclin, heparin, salicylates, thrombocytic agents,anti-proliferative agents, nitrates, calcium channel blocking drugs,streptokinase, urokinase, tissue plasminogen activator (TPA) andanisoylated plasminogen activator (PA) and anisoylatedplasminogen-streptokinase activator complex (APSAC), colchicine andalkylating agents, growth modulating factors such as interleukins,transformation growth factor P and congeners of platelet derived growthfactor, monoclonal antibodies directed against growth factors, modifiedextracellular matrix components or their receptors, lipid andcholesterol sequestrants and other agents which may modulate vesseltone, function, arteriosclerosis, and the healing response to vessel ororgan injury post intervention.

Foaming Agent.

In certain embodiments, the substrate may include a foaming agent which,upon combination with the crosslinker composition, results in a foamingcomposition, e.g., a composition that includes gaseous air bubblesinterspersed about. Any convenient foaming agent may be present, wherethe foaming agent may be an agent that, upon contact with thecrosslinking composition, produces a gas that provides bubble generationand, hence, the desired foaming characteristics of the composition. Forexample, a salt such as sodium bicarbonate in an amount ranging fromabout 2 to about 5% w/w may be present in the substrate. Uponcombination of the substrate with an acidic crosslinker composition,e.g., having a pH of about 5, a foaming composition is produced.

Additional Modifiers.

Additional modifiers may also be present. For example, blends of one ormore polymers (e.g., polyblends), such as Teflon, PET, NYLON, hydrogels,polypropylene, etc., may be present. The polyblends may be modified,e.g., as described above, to provide for desired properties. Theseadditional modifiers may be present in amounts ranging from about 0 to50%, including from about 10 to about 30%.

Crosslinker Composition.

As indicated above, the phase invertible composition is produced bycombining a proteinaceous substrate, as described above, with acrosslinker, where the crosslinker stabilizes the proteinaceoussubstrate, e.g., by forming covalent bonds between functionalitiespresent on different polypeptide strands of the proteinaceous substrate.Crosslinking typically renders the molecules of the composition lesssusceptible to chemical degradation, and as such modifies the resorptioncharacteristics of the composition as well as the biological responsesinduced by the presence of the composition. Numerous crosslinking agentshave been identified. Examples of crosslinking agents include, but arenot limited to: photo-oxidative molecules; carbodimides; carbonylcontaining compounds, e.g., mono- and dicarbonyls, including carboxilicacids, e.g., dicarboxylic acids, such as adipic acid, glutaric acid andthe like, and aldehydes, including mono- and dialdehydes, e.g.glutaraldehyde; etc. In certain embodiments, the crosslinker employed isan aldehyde crosslinker. In certain of these embodiments, the aldehydecrosslinker is pretreated to produce a stabilized aldehyde crosslinker,for example, a stabilized glutarhaldehyde crosslinker, e.g., where thecrosslinker is heat stabilized aldehyde, such as heat stabilizedglutaraldehyde (such as described in U.S. Pat. No. 7,303,757, thedisclosure of which is herein incorporated by reference).

The amount of crosslinking agent in the composition may vary. In certainembodiments the amount of crosslinking agent ranges from 0.1 to 20%(v/v), such as 0.5 to 15% (v/v) and including 1 to 10% (v/v).

In many embodiments, the phase invertible composition is produced bycombining a liquid proteinaceous substrate, as described above, with aliquid crosslinker composition that includes a macromolecularcrosslinking agent. The macromolecular crosslinking agent is produced bycombining an excess of a crosslinking agent, e.g., a heat-treateddialdehyde, with an amount of a physiologically acceptable polymer, suchas a glycosaminoglycan. The excess of crosslinking agent andphysiologically acceptable polymer react to produce a macromolecularcrosslinking agent. Upon combination of the macromolecular crosslinkingagent with the proteinaceous substrate, the macromolecular crosslinkingagent reacts with proteins in the substrate component to produce a finalcomposition characterized by the presence of an interpenetratingnetwork.

As such, the macromolecular crosslinking agent of the crosslinkercomponent is one that is produced by combining an excess of acrosslinking agent with an amount of a physiologically acceptablepolymer. In this embodiment, the crosslinking agent is present in thecrosslinking composition in excess with respect to the amount ofphysiologically acceptable polymer. In certain embodiments the amount ofcrosslinking agent ranges from 0.1 to 20% (v/v), such as 0.5 to 15%(v/v) and including 1 to 10% (v/v). In certain embodiments, the amountof physiologically acceptable polymer makes up from 0.01 to 5% (wt/v),such as 0.05 to 3% (wt/v) including 0.1 to 1% (w/v) of the crosslinkingcomposition.

The physiologically acceptable polymer may be any polymer that istolerated by the body and reacts with the crosslinking agent to producea prepolymer macromolecular crosslinking product. The prepolymer productis one that is a reaction product between the crosslinking agent and thepolymer, and includes a polymer backbone with one or more crosslinkingmolecules covalently bonded thereto such that the polymer backboneincludes one or more crosslinking functional groups, where thecrosslinking functional groups include at least one reactive moiety,e.g., an aldehyde moiety, that can covalently bond to the proteincomponent of the proteinaceous substrate. As such, the prepolymerproduct retains the ability to crosslink upon contact with theproteinaceous substrate. The prepolymer product of certain embodimentsis a soluble macromolecule that comprises a polymeric backbone moleculebound to crosslinking agent molecules, where at least a portion of thebound crosslinking agent molecules retain a free crosslinking moietythat can bind proteins in the proteinaceous substrate. This prepolymerproduct “macromolecular” crosslinking agent may vary in averagemolecular weight, and in certain embodiments may range in weight from10,000 to 4 million Daltons, such as 500,000 to 2 million Daltons.

In certain embodiments, the physiologically acceptable polymer is aglycosaminoglycan (i.e., a mucopolysaccharide). Specificglycosaminoglycans of interest include, but are not limited to:chondroitin sulphate; dermatan sulphate; keratan sulphate; heparin;heparan sulphate; and hyaluronan (i.e., hyaluronic acid). In certainembodiments, the glycosaminoglycan component is hyaluronan.

The crosslinking component, such as those described above, may besterilized according to any convenient protocol, where sterilizationprotocols of interest include, but are not limited to: gamma radiation,electron beam radiation, and the like. Generally, the liquidcrosslinking component is one that is storage stable. By storage stableis meant that the substrate may be maintained under storage conditions,such as room temperature for a period of time of at least 3 years orlonger, such as 5 years or longer, without undergoing any substantialchange that negatively impacts the function of the substrate such thatit is no longer suitable for use in preparing a biocompatible phaseinvertible composition of the invention.

While the viscosity of the crosslinking component may vary, in certainembodiments it approximates the viscosity of the proteinaceouscomponent, expressed in centistokes (cSt) at about 25. degree. C.,ranging from about 10 cSt to 150 cSt, such as about 30 cSt to 70 cSt. Incertain embodiments, the viscosity of the crosslinker compositionapproximates the viscosity of the proteinaceous substrate composition,expressed in centistokes (cSt) at about 25. degree. C., such as rangingfrom about 10 cSt to 200 cSt, including from about 20 cSt to 150 cSt,and typically about 30 cSt to 125 cSt.

In certain embodiments, the crosslinker composition may further includean amount of a viscosity modifying agent. Viscosity modifying agents ofinterest include, but are not limited to: polyoxyethylene orpolyoxypropylene polymers or copolymers thereof, such as polyethyleneglycol and polypropylene glycol; nonionic cellulose ethers such asmethylcellulose, ethylcellulose, hydroxymethylcellulose,hydroxyethylcellulose, carboxymethylcellulose, carboxyethylcellulose andhydroxypropylcellulose; additional celluloses, such ascarboxymethylcellulose sodium, carboxymethylcellulose calcium,carboxymethylstarch; and the like. In certain embodiments of particularinterest, the emulsifying agent is a cellulose ether, particularly anonionic cellulose ether, such as carboxymethylcellulose.Carboxymethylcellulose is available from a variety of commercialsources, including but limited to, Sigma, Hercules, Fluka and Noviant.In certain embodiments, the average molecular weight of the celluloseether is at least about 1000 Daltons, such as at least about 5000Daltons, where the average molecular weight may be as high as 10,000Daltons or higher, e.g., 50,000 Daltons or higher, 100,000 Daltons orhigher, and ranges in certain embodiments from about 5,000 to about100,000 Daltons, such as from about 10,000 to about 50,000 Daltons. Theproportion of the viscosity modifying agent in the sealant in certainembodiments ranges from 0.01 to 10% (v/v), such as 0.1 to 4% (v/v)including 0.5 to 2% (v/v).

Buffer.

Upon mixture of the proteinaceous substrate and crosslinker to producethe subject phase invertible composition, buffering of the phaseinvertible composition is employed in certain embodiments for a numberof reasons, e.g., to optimize the bonding strength of the composition tothe attaching surface, to optimize the conditions necessary for internalcrosslinking to occur, etc. For example, optimum crosslinking forproteins using glutaraldehyde crosslinkers occurs at pH range from about6 to about 8. Buffers capable of maintaining this range are useful inthis invention, as long as they do not interfere with the carbonylterminal of the crosslinker or modify the amine terminus of the aminoacids. For example, phosphate buffers have a pKa value in the range ofpH 7.0 and do not interfere with the crosslinking process because theydo not contain carboxylic or amine functionalities. Phosphate buffer upto 1M in strength is suitable for use as a buffer in the presentinvention, where in certain embodiments the phosphate buffer is about0.01 to about 0.3M in strength. While phosphate buffering of thesolutions is ideal for the stability of the protein substrate inapplications where increased adhesion is required, an acidic buffer maybe used as well. Citrate buffers 0.1-1 M and having a pH range of about4.5 to about 6.5 have been found to be useful for this invention.

The buffer may be present in either the initial crosslinker component orthe initial proteinaceous substrate component, or present in bothcomponents, as desired.

Combination of Substrate and Crosslinker to Produce Phase InvertibleComposition.

As summarized above, the subject phase invertible compositions areprepared by combining a liquid proteinaceous substrate and a liquidcrosslinker in appropriate amounts and under conditions sufficient forthe phase invertible composition to be produced. In certain embodiments,the substrate and crosslinker are combined in a ratio (v/v) ranging fromabout 1/5 to about 5/1; so that a resultant phase invertible compositionis produced in which the total protein concentration ranges from about10 to about 60%, such as from about 20 to about 50%, including fromabout 30 to about 40% and the total crosslinker composition ranges fromabout 0.1 to about 20%, such as from about 0.5 to about 15%, includingfrom about 1 to about 10%.

Combination of the substrate and crosslinker typically occurs undermixing conditions, such that the two liquid components are thoroughlycombined or mixed with each other. Combination or mixing may be carriedout using any convenient protocol, e.g., by manually combining twocomponents, by employing a device that combines the two components, etc.Combination or mixing is typically carried out at a temperature rangingfrom about 20 to about 40. degree. C., such as room temperature.

Combination of the proteinaceous substrate and crosslinker as describedabove results in the production of a phase invertible composition. By“phase invertible composition” is meant a composition that goes from afirst liquid state to a second non-fluid, e.g., gel or solid, state. Inthe second non-fluid state, the composition is substantially, if notcompletely, incapable of fluid flow. The phase invertible compositiontypically remains in a fluid state, following combination of thesubstrate and crosslinker components, for a period of time ranging fromabout 10 seconds to about 10 minutes, such as from about 20 seconds toabout 5 minutes, including from about 30 seconds to about 120 second,when maintained at a temperature ranging from about 15. degree. C. toabout 40. degree. C., such as from about 20. degree. C. to about 30.degree. C.

Specific phase invertible formulations include as components: (i) afirst composition comprising a proteinaceous substrate, the substratecomposition comprising a branched polyethylenimine, about 30%-50%albumin, and about 0.1%-0.3% chitosan, wherein the weight ratio ofbranched polyethylenimine to albumin is about 1:5 to about 1:80; and(ii) a second composition comprising a crosslinking composition havingabout 3%-10% heat treated glutaraldehyde, and optionally, one or more ofabout 0.1%4% hyaluronic acid, and about 0%-1.5% sodium salt ofcarboxymethylcellulose high viscosity.

In a particular embodiment, a phase invertible formulation is providedthat includes as components: (i) a first composition comprising aproteinaceous substrate composition having a branched polyethylenimine,about 40% albumin, and about 0.16% chitosan, wherein the weight ratio ofbranched polyethylenimine to albumin is about 1:10 to about 1:40; and(ii) a second composition comprising a crosslinking composition havingabout 4.4%-7.5% heat treated glutaraldehyde, and optionally, one or moreof about 0.2% hyaluronic acid, and about 0%-0.75% sodium salt ofcarboxymethylcellulose high viscosity.

As also described above, various additional materials can beincorporated into the phase invertible compositions to serve any ofseveral purposes, such as to modify the physical characteristics of thecomposition, and/or aid in the repair of a target tissue or biologicalmaterial to which the composition is applied. For example, biologicallyactive agents such as peptides, proteins, nucleic acids, carbohydratemolecules, small molecules and the like can be incorporated to attractand bind specific cell types, such as white cells and platelets, ormaterials such as fibronectin, vimentin, and collagen, can be used toenhance healing by non-specific binding. Tracing material, such asbarium, iodine or tantalum salts, may also be included to allowvisualization and/or monitoring of the phase invertible composition. Incertain embodiments, different biologically active agents can be used indifferent applications or layers when one or more phase invertiblecompositions is differentially applied.

In certain embodiments, cells can also be incorporated into or appliedin connection with the phase invertible compositions before, duringand/or after application of the composition. When employed, cells aregenerally included in the proteinaceous substrate composition, orapplied in conjunction with or separately from the phase invertiblecomposition in a manner that allows their adherence at the site ofapplication. The cells can be living, artificial cells, cell ghosts(i.e., red blood cell or platelet ghosts), or pseudovirions, dependingon a given end use. For example, the cells may be selected to producespecific agents such as growth factors at the site of application.Biologically active agents that modulate cell viability,differentiation, and/or growth can be included as well. In someembodiments, the cells may be stem cells, or in other embodiments,progenitor cells corresponding to the type of tissue at the treatmentlocation or other cells, providing therapeutic advantages. For example,liver cells incorporated into the phase invertible composition andapplied to a wound or surface of the liver of a patient may aid in itsregeneration and repair. This may be particular useful in cases wherediseases such as cirrhosis, fibrosis, cystic disease or malignancyresults in non-functional tissue, scar formation or tissue replacementwith cancerous cells. Similar methods may be applied to other organs andtissues as well.

The biologically active agents can be incorporated physically and/or bychemical attachment. Physical incorporation is carried out by mixing thebiologically active agent with the phase invertible material prior toand/or during application to the target surface and curing. The materialis usually mixed into the proteinaceous substrate solution to form asolution, suspension or dispersion. In one embodiment, the biologicallyactive agent can be encapsulated within delivery devices such asmicrospheres, microcapsules, liposomes, cell ghosts or pseudovirions,which in themselves effect release rates and uptake by cells. Chemicalincorporation of the biologically active agent is carried out bychemically coupling the agent to a polymeric material of either theproteinaceous substrate or crosslinking composition, usually theproteinaceous substrate, before or at the time of polymerization (e.g.,by conjugation through reactive functional groups, such as amines,hydroxyls, thiols, and the like). To ensure that the desired cure timesand burst properties of the phase invertible composition are maintained,physical and/or chemical incorporation of a biologically active agenttakes into consideration the relative amounts of proteinaceous substrateand crosslinker present in the final composition, and can be adjustedaccordingly.

Methods.

The subject biocompatible phase invertible compositions are employed inmethods where a quantity of the phase invertible composition isdelivered to a particular site or location of a subject, patient or hostin need thereof, typically a surgical site, injury site, suturinglocation, or other location with bleeding or exposed blood. The subject,patient or host is typically a “mammal” or “mammalian,” where theseterms are used broadly to describe organisms which are within the classmammalian, including, but not limited to, the orders carnivore (e.g.,dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), lagomorpha(e.g. rabbits) and primates (e.g., humans, chimpanzees, and monkeys). Inmany embodiments, the animals or hosts, i.e., subjects (also referred toherein as patients) will be humans.

The quantity that is delivered to the subject in any given applicationwill necessarily vary depending on the nature of the application and useof the composition, but in certain representative embodiments rangesfrom about 1 to about 50 ml, such as from about 1 to about 25 ml,including from about 1 to about 5 ml, e.g., about 3 ml.

While necessarily dependent on the particular application in which thesubject composition is being employed, the subject composition is, inmany embodiments, locally delivered to a particular region, site orlocation of the host, where the site or location may, of course, vary.Representative sites or locations include, but are not limited to:vessels, organs, and the like. Depending on the particular application,the composition may be delivered to the site of interest manually orwith a delivery device, e.g., the delivery device employed to deliverthe composition in stenting applications, described in greater detailbelow.

Utility.

The subject biocompatible phase invertible compositions find use in avariety of different applications. Representative applications of thesubject phase invertible compositions include those described in U.S.Pat. Nos. 3,438,374; 5,092,841; 5,292,362; 5,385,606; 5,575,815;5,583,114; 5,843,156; 6,162,241; 6,290,729; 6,302,898; 6,310,036;6,329,337; 6,371,975; 6,372,229; 6,423,333; 6,458,147; 6,475,182;6,547,806; and 7,303,757; as well as U.S. Application Nos. 2002/0015724;2002/0022588; 2002/0133193; 2002/0173770; 2002/0183244; 2002/019490;2002/0032143; the disclosures of which are herein incorporated byreference.

Systems.

Also provided are systems for use in practicing the subject methods. Thesystems may include fluid delivery elements for delivery of thesubstrate and crosslinking composition to the site of administration,mixing elements, etc. Examples of such systems include those describedin U.S. Pat. No. 7,303,757; the disclosure of which is hereinincorporated by reference.

Kits.

Also provided are kits for use in practicing the subject methods, wherethe kits typically include a distinct liquid substrate and liquidcrosslinking composition components of a phase invertible fluidcomposition, as described above. The substrate and crosslinkingcompositions may be present in separate containers in the kit, e.g.,where the substrate is present in a first container and the crosslinkingagent is present in a second container, where the containers may or maynot be present in a combined configuration.

The subject kits may also include a mixing device, for mixing thesubstrate and crosslinker together to produce the phase invertiblecomposition. The kits may also include a delivery device (which may ormay not include a mixing element), such as a dual barrel syringe,catheter devices, and the like, as described above.

The kit may further include other components, e.g., guidewires, sensorwires, etc., which may find use in practicing the subject methods.

In addition to above-mentioned components, the subject kits typicallyfurther include instructions for using the components of the kit topractice the subject methods. The instructions for practicing thesubject methods are generally recorded on a suitable recording medium.For example, the instructions may be printed on a substrate, such aspaper or plastic, etc. As such, the instructions may be present in thekits as a package insert, in the labeling of the container of the kit orcomponents thereof (i.e., associated with the packaging or subpackaging)etc. In other embodiments, the instructions are present as an electronicstorage data file present on a suitable computer readable storagemedium, e.g. CD-ROM, diskette, etc. In yet other embodiments, the actualinstructions are not present in the kit, but means for obtaining theinstructions from a remote source, e.g. via the Internet, are provided.An example of this embodiment is a kit that includes a web address wherethe instructions can be viewed and/or from which the instructions can bedownloaded. As with the instructions, this means for obtaining theinstructions is recorded on a suitable substrate.

The following examples are provided by way of illustration and not byway of limitation.

Experimental

Various formulations of phase invertible compositions were prepared inwhich branched polyethylenimine (PEI) [50,000 to 100,000 molecularweight] was included in the protein component. Illustrative formulationswere tested as shown in Table 1 in which the viscometer used was405-U190, the temperature was 23.7. degree. C. (room temperature), andthe viscosity constant was 2.528 cSt/s (centistokes per second).

TABLE 1 Hydrogel Study-Viscosity Measurement Time Kinematic SampleAlbumin (%) Chitosan (%) PEI (%) Travel (s) Viscosity (cSt) 1 40 0.16  0 7  16.853 2 40 0.16  1 28  70.784 3 40 0.16  2 41 102.805 4 40 0.16  474 187.072 5  0 0   20  4  10.112 Data obtained from the average of N =3 Kinematic Viscosity = Time Travel (s) × Viscosity Constant (cSt/s)Key: PEI = polyethylenimine; s = seconds; cSt = centistokes

The above formulations were tested before combining with thecross-linker solution.

The results in shown in FIG. 1 and Table 1 demonstrate that when apolyamine such as a polyethylenimine is provided in the proteincomponent at particular weight ratios, the viscosity is synergisticallyenhanced, which results in an improved overall two component sealantcomposition.

It is evident from the above results and discussion that the presentinvention provides an important new type of biocompatible compositionthat can be used in a variety of different applications, where benefitsof the subject compositions include, but are not limited to, highadhesion, low toxicity, and the like. Accordingly, the present inventionrepresents a significant contribution to the art.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart, in light of the teachings of this invention, that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims.

What is claimed is:
 1. A phase invertible composition produced bycombining: a substrate comprising a polyamine and a proteinaceousmaterial in a weight ratio of polyamine to proteinaceous material beingin the range from 1:10 to 1:40, wherein the combination of the polyamineand the proteinaceous material has a viscosity which is greater thanthat of either the polyamine or the proteinaceous material alone; and acrosslinking agent, wherein the crosslinking agent causes the polyamineand the proteinaceous material to crosslink and the polyamine and theproteinaceous material crosslink in the presence of the crosslinkingagent, wherein the crosslinking agent is exposed to radiation, whereinthe substrate has a kinematic viscosity of about 70 to 200 centistokes,wherein the proteinaceous material is selected from the group consistingof albumin, elastin, fibrin and soluble and insoluble forms of collagenand combinations thereof, and wherein the crosslinking agent comprises amacromolecular crosslinking agent comprising a reaction product of anexcess of a liquid aldehyde cross linking agent and a glycosaminoglycan.2. A kit for producing a phase invertible composition, the kitcomprising: a substrate comprising a polyamine and a proteinaceousmaterial in a weight ratio which produces a synergistic viscosityenhancing effect when combined, the weight ratio of polyamine toproteinaceous material being in the range from 1:10 to 1:40, wherein thecombination of the polyamine and the proteinaceous material has aviscosity which is greater than that of either the polyamine or theproteinaceous material alone; and a crosslinking agent compositioncomprising a crosslinking agent, wherein the crosslinking agent causesthe polyamine and the proteinaceous material to crosslink and thepolyamine and the proteinaceous material crosslink in the presence ofthe crosslinking agent, wherein the crosslinking agent is exposed toradiation, wherein the substrate has a kinematic viscosity of about 70to 200 centistokes, wherein the proteinaceous material is selected fromthe group consisting of albumin, elastin, fibrin and soluble andinsoluble forms of collagen and combinations thereof and wherein thecrosslinking agent comprises a macromolecular crosslinking agentcomprising a reaction product of an excess of a liquid aldehyde crosslinking agent and a glycosaminoglycan.
 3. The kit of claim 2, whereinthe kit further comprises a phase invertible fluid delivery device. 4.The kit of claim 2, wherein the radiation sterilizes the crosslinkingagent.
 5. The kit of claim 2, wherein the radiation comprises gammaradiation.
 6. The kit of claim 2, wherein the radiation compriseselectron beam radiation.
 7. The kit of claim 2, wherein the polyamine isa polyalkyleneimine.
 8. The kit of claim 7, wherein thepolyalkyleneimine is a polyethyleneimine.
 9. The kit of claim 8, whereinthe polyethyleneimine is a branched polyethyleneimine.
 10. The kit ofclaim 2, wherein the substrate further comprises a carbohydrate.
 11. Thekit of claim 10, wherein the carbohydrate is chitosan.
 12. The kit ofclaim 2, wherein the crosslinking agent comprises an aldehyde.
 13. Thekit of claim 12, wherein the aldehyde is glutaraldehyde.
 14. The kit ofclaim 13, wherein the glutaraldehyde is heat stabilized.
 15. The kit ofclaim 2, wherein the glycosaminoglycan is hyaluronan.
 16. The kit ofclaim 2, wherein the viscosity of the substrate approximates that of thecrosslinking agent.
 17. The kit of claim 16, wherein the crosslinkingagent further comprises a viscosity modifying agent.
 18. The kit ofclaim 2, wherein the substrate further comprises an agent selected fromthe group consisting of a tackifying agent, and a plasticizing agent.19. The kit of claim 2, wherein the crosslinking agent further comprisesan agent selected from the group consisting of a tackifying agent, and aplasticizing agent.
 20. The kit of claim 2, wherein the substratecomprises about 1 to about 4% (w/w) branched polyethyleneimine, about30% to about 50% albumin, and about 0.1% to about 0.3% chitosan, and thecrosslinking agent comprises a macromolecular crosslinking agentcomprising a reaction product of about 3% to about 10% heat-treatedglutaraldehyde and a glycosaminoglycan.
 21. The kit of claim 2, whereinthe a substrate comprises about 1 to about 4% (w/w) branchedpolyethyleneimine, about 40% albumin, and about 0.16% chitosan, and thecrosslinking agent comprises a macromolecular crosslinking agentcomprising a reaction product of about 4.4% to about 7.5% heat-treatedglutaraldehyde and a glycosaminoglycan.